The most abundant member of the alpha acid family is (−)-humulone. The absolute configuration of (−)-humulone has been reported previously (De Keukeleire 1970), as well as the absolute configurational assignments of cis and trans iso-alpha acids which are derived from (−)-humulone (De Keukeleire 1971).
The so-called ‘reduced iso-alpha acids’—reduction products derived from both the cis and trans iso-alpha acids—are further categorized. This additional categorization depends on the location and degree of saturation. The class of compounds resulting from the reduction of only the C6 carbonyl to a hydroxyl is collectively referred to as the rho iso-alpha acids (RIAAs). The tetrahydro iso-alpha acid (THIAA) class refers collectively to those compounds where only both isoprenyl moieties are saturated. Similarly, the hexahydro-iso-alpha acids (HIAAs) refers collectively to reduced derivatives of iso-alpha acids containing both the hydroxyl at C6 and saturation of both isoprenyl moieties. In addition to the existence of cis and trans diastereomers, there are a variety of congeners within each of the three classes as a result of the incorporation of various short-chain fatty acids into the biosynthetic pathway of the corresponding phlorglucinols (Wang 2008). The phlorglucinols are common precursors to the alpha acids that are precursors to iso-alpha acids, which are in turn precursors to the reduced alpha iso-acids.
Recently the reduced iso-alpha acids have been shown to possess beneficial effects in the treatment of obesity, dyslipidemia, and inflammation in a variety of in vitro and murine models. A mixture of congeners and stereoisomers of rho iso-alpha acids—referred to as RIAA—demonstrated anti-inflammatory activity in tumor necrosis factor alpha (TNF-α)-stimulated mature 3T3-L1 adipocytes and lipogenic activity in differentiating 3T3-L1 adipocytes (Babish 2010).
A mixture of congeners and stereoisomers of tetrahydro iso-alpha acids—referred to as THIAA—inhibited the activities of spleen tyrosine kinase (Syk), Bruton's tyrosine kinase (Btk), phosphatidylinositol 3-kinase (PI 3-kinase), and glycogen synthase kinase 3β (GSK3β) as well as β-catenin phosphorylation in vitro. Additionally, THIAA inhibited osteoclastogenesis, as indicated by decreased transformation of RAW 264.7 cells to osteoclasts and reduced TRAP activity, and inhibited IL-1β-activated prostaglandin E2, matrix metalloproteinase 3, IL-6, IL-8, and monocyte chemotactic protein 1 in RASFs. Furthermore, in mice with collagen induced arthritis (CIA), this same mixture of congeners and stereoisomers (i.e., THIAA) significantly reduced the arthritis index and decreased bone, joint, and cartilage degradation. Serum IL-6 concentrations in these mice were also inhibited in a dose-dependent manner when treated with THIAA (Konda 2010).
Consistent with these findings it has also been reported that RIAA and THIAA, respectively, inhibited prostaglandin E2 (PGE2) production in lipopolysaccharide-stimulated RAW 264.7 macrophages as well as inhibited inducible cyclooxygenase-2 (COX-2) protein expression. In addition to this, each of these mixtures, i.e., RIAA and THIAA, respectively, are reported to have reduced NF-κB nuclear translocation and abundance in a dose dependent manner (Tripp 2009).
The heterogeneity of the RIAA and THIAA prevents an understanding or knowledge of the relationship among the various congeners and stereo-isomers—present in either the RIAA or THIAA mixture—with respect to their individual and hence relative biological activities. Furthermore, the potential for synergy among the numerous compounds present in RIAA and THIAA may account for much, if not all, of the observed biological activity of these mixtures.
In order to achieve an understanding and knowledge of the relationship among the various congeners and stereo-isomers with respect to their individual and hence relative biological activity, it is imperative that the compounds of interest are obtained, as substantially and enantiomerically pure compounds, and that their individual biological activity is measured in a substantially and enantiomerically pure form. This imperative necessarily follows from the dependence of the biological activity of any heterogenous substance comprised of a mixture of different molecules (e.g., THIAA, RIAA, and HHIAA) on the percent composition, stereochemistry, structure and other properties of the different molecules that comprise the heterogeneous substance. For these reasons there exists a need to prepare and purify substantially enantiomerically pure reduced iso-alpha acid derivatives for use in pharmaceutical compositions and treatments.
In addition to this need there is also a need to manufacture a substantially and enantiomerically pure compound in a form that is suitable for various processes routinely encountered in pharmaceutical development. The manufacture of pure crystalline forms of potential drug candidates is advantageous for drug development. One advantage is the improved characterization of a drug candidate's chemical and physical properties. It is not uncommon for crystalline forms to possess more favorable pharmacokinetics compared to an amorphous form and they are often easier to process. Improved storage stability is an additional advantage often associated with crystalline forms. The physical properties inherent to crystalline forms of potential drug candidates are a significant factor in choosing a particular pharmaceutical active ingredient. An example is the formulation of the potential drug into a suitable composition for manufacture, storage and consumption. Specifically, the flowability of the crystalline solid, pre- and post-milling greatly impacts the manner of how the drug candidate is handled during processing and dose manufacture. In cases where particles of the milled solid form do not flow easily, formulations scientists will endeavor to develop a formulation to compensate for this difficulty. This often involves the use of glidants such as colloidal silicon dioxide, talc, and starch or tri-basic calcium phosphate. An additional solid-state property of a potential pharmaceutical compound is its dissolution rate in aqueous fluid. The physical properties associated with a particular crystalline polymorph are inherently due to the spatial orientation and unique conformation of individual molecules that comprise the unit cell of the crystalline polymorph. A particular crystalline polymorph possesses unique thermal properties that are generally different from either an amorphous form or another polymorph.
The thermal properties of polymorphs are measured using techniques such as capillary melting point, thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). The results of these measurements are used to distinguish and identify the existence of polymorphic forms and distinguish them from each other. A particular polymorphic form generally possesses distinct crystallographic and spectroscopic properties that are detectable by a variety of techniques including but not limited to powder X-ray powder diffraction (XRPD), single crystal x-ray crystallography, and infrared spectrometry.